Doi:10.1016/j.procbio.2005.11.024

Process Biochemistry 41 (2006) 1217–1222 Antiproliferative activity of fish protein hydrolysates on L. Picot S. Bordenave S. Didelot I. Fruitier-Arnaudin F. Sannier G. Thorkelsson J.P. Berge´ , F. Gue´rard A. Chabeaud J.M. Piot a Laboratoire de Biotechnologies et Chimie Bio-organique (LBCB), FRE 2766-CNRS, UFR Sciences Fondamentales et Sciences pour l’Inge´nieur, Baˆtiment Marie Curie, Universite´ de la Rochelle, b Icelandic Fisheries Laboratories (IFL) and University of Iceland, Skulagata 4, IS-101 Reykjavik, Iceland c IFREMER Centre de Nantes, De´partement Sciences et Techniques Alimentaires, BP 1105, F-44311 Nantes Cedex, France d Laboratoire ANTIOX, Universite´ de Bretagne Occidentale, Poˆle Universitaire Pierre-Jakez He´lias, 18 avenue de la plage des gueux, F-29018 Quimper Cedex, France Received 2 June 2005; received in revised form 29 November 2005; accepted 29 November 2005 Antiproliferative activity of 18 fish protein hydrolysates was measured on 2 human breast cancer cell lines grown in vitro. Three blue whiting, three cod, three plaice and one salmon hydrolysates were identified as significant growth inhibitors on the two cancer cell lines. Preliminaryanalysis of hydrolysates composition evidenced they contained a complex mixture of free amino acids, peptides with various sizes ranging up to7 kDa and in a lower proportion, lipids and sodium chloride. RP-HPLC fractionation of fish hydrolysates is currently undertaken to purifyanticancer peptides, lipids or other bioactive trace compounds responsible for this antiproliferative activity.
# 2005 Elsevier Ltd. All rights reserved.
Keywords: Breast cancer; Fish protein hydrolysate (FPH); Antiproliferative activity enrichment of fillets. Fish protein hydrolysates (FPH),obtained by controlled enzymatic hydrolysis, are among Fish consumption is associated to health benefits because the best protein hydrolysates in term of nutritional properties of a rich content in proteins of high nutritional value, (balanced amino acid composition, high digestibility , but minerals, vitamins and distinctive lipids . Estimates for are mainly used for animal nutrition because of their bitter 2003, based on reports from major fishing countries, indicate that fish provided more than 2.6 billion people with at least The identification of growth factors , immunomodulating 20% of their average animal protein intake . However, fish antimicrobial antithrombotic hypotensive , and stocks are decreasing and the annual world fish catch is anticancer peptides in hydrolysates from milk , stabilized. Moreover, a rational valorization of by-products is extensively investigated, as the yearly average discards are evidence that enzymatic hydrolysis of food proteins is an estimated to 7.3 million tonnes Fish proteins show efficient way to recover potent bioactive peptides. Also, interesting rheological properties and can be valorized for research on fish or FPH demonstrated they constitute a source numerous applications such as production of surimi or protein of promising health benefits molecules for nutritional orpharmaceutical applications Isolation of potentanticancer compounds from fish tissue is also a strongargument to consider that by-products could constitute a source Abbreviations: Ara-C, cytosine-beta-D-arabinofuranoside; DAD, of anticancer or chemopreventive molecules.
diode array detector; FPH, fish protein hydrolysate; SEC, size In this context, a preliminary study was designed to determine whether FPH exert an antiproliferative activity on * Corresponding author. Tel.: +33 5 46 45 82 20; fax: +33 5 46 45 82 65.
E-mail address: [email protected] (L. Picot).
1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.11.024 L. Picot et al. / Process Biochemistry 41 (2006) 1217–1222 pipetted into separate columns of the microplate and equilibrated at 37 8C.
Cancer cells grown in flask to confluence were rinsed three times with PBS at 37 8C, trypsinized, and centrifugated (3000 Â g, 3 min). Cells were washedwith fresh medium and resuspended in culture medium at a concentration of105 cells mLÀ1. The cell suspension (50 mL) was then added to each well to Twenty-one FPH were prepared by three European fish processing com- deposit 5000 cells and adjust the final fish hydrolysate (1 g LÀ1) or SDS (1%) panies: Coope´rative de Traitement des Produits de la Peˆche (CTPP), Boulogne/ concentrations. The microplate was then incubated at 37 8C for 72 h. At the end mer, F-62203-France; Marinova (Danish Fish Protein), Hoejmark, DK-6940- of the incubation, 15 mL of MTS (soluble tetrazolium salt) solution was added Denmark and Primex, Reykjavik, IS-101-Iceland, from Atlantic salmon (Salmo to each well, and the plate was incubated for a further 4 h to allow MTS salar), Atlantic cod (Gadus morhua), plaice (Pleuronectes platessa), blue metabolism to formazan by the succinate-tetrazolium reductase only active in whiting (Micromesistius poutassou), Atlantic emperor (Lethrinus atlanticus), viable cells. A solubilization/stop solution (100 mL) was added to stop the pollack (Pollachius pollachius) and Portuguese dogfish or siki (Centroscymnus succinate-tetrazolium reductase activity, kill the cells and solubilize formazan cristals for 12 h at 37 8C. Optical densities were read on a plate reader(VERSAmax, Molecular Devices France, 35762 Saint Gre´goire) at 570 nm.
2.2. Fish protein hydrolysates preparation and analysis The data were analyzed to calculate the percentage of growth inhibition inducedby the presence of FPH in cell culture medium determined by the equation: Fish proteins were purified from fresh filleting by-products or headed and gutted by-catches using the pH-shift extraction method based on the solubility of myofibrillar and sarcoplasmic proteins at extreme acid and alkaline pH. Fish muscle was minced, prewashed with water and centrifuged (3000 Â g,10 min). The resulting pellet was dissolved in water (1:6, w:v) and pH was where ODFPH, ODlysis and ODcontrol are optical densities, respectively measured adjusted with NaOH 1 M at 10.8, to solubilize myofibrillar and sarcoplasmic for cells grown in culture medium containing FPH 1 g LÀ1, SDS 1% or PBS only.
proteins. Proteins were then efficiently separated from lipids, membranes, skin Antiproliferative activity of FPH was compared with that of reference anticancer and bones by centrifugation (3000 Â g, 10 min). Two phases were obtained: a molecules (Sigma–Aldrich) with various cellular targets, namely actinomycine D solid phase containing membranes, skin and bones (pellet) and a soluble phase (intercalating agent), Ara-C (nucleoside analogue), cyclophosphamide (alkylat- containing solubilized proteins. The soluble phase was carefully collected and ing agent), etoposide (topoisomerase II inhibitor), kenpaullone and roscovitine pH was adjusted to 5.6 with HCl 1 M, to precipitate proteins, which were (cyclin-dependent-kinases inhibitors). Anticancer compounds were dissolved in collected by centrifugation (3000 Â g, 10 min). The pellet was dissolved in two DMSO to give 10À3 M stock solutions from which further dilutions were made in parts water at pH 7.5 and hydrolyzed at 55–57 8C for 106 min with Protamex culture medium to get final concentrations of 10À9 and 10À6 M. For control 98 g/1000 kg and Alcalase 20 mL/1000 kg (Novo Nordisk). The enzymes were experiments, culture cell medium contained a DMSO concentration equivalent to inactivated by heating the resulting suspension to 90 8C for 10 min. The that found in the 10À6 M anticancer molecule assay.
suspension was centrifuged (3000 Â g, 10 min) to pellet inactivated proteasesand non-hydrolyzed fish proteins. The supernatant, containing soluble peptides, Peptide, lipid and sodium chloride content of FPH were analyzed by the Antiproliferative data are given as mean percentage Æ S.E.M of 24 assays certificated laboratory IFL, 101-Reykjavik, Iceland, following the Kjeldahl from triplicate independent experiments. Statistical significance of growth (ISO 5983), Volhard (AOAC 937-09) and Soxhlet methods (AOCS Official inhibition induced by FPH was calculated following x2 test with a = 0.05.
Method), respectively. Molecular weight distributions of FPH peptides weredetermined using SEC in FPLC mode on a Superdex Peptide HR 10/30 column(Pharmacia, fractionation range: 7000–100 Da) with a Waters 600 automated gradient controller pump and a Waters 996 DAD. The mobile phase (isocratic)consisted of MilliQ water-TFA 0.1% and acetonitrile (70:30) at a flow rate of 0.5 mL minÀ1. Hydrolysates were dissolved in mobile phase to 5 g LÀ1 andsterile filtered. Sample size was 40 mL (200 mg of hydrolysate) and peptideswere detected at 220 nm. Millenium software was used to collect, plot and shows the composition of FPH. Most hydrolysates process the chromatographic data. Standard peptides (Sigma, France) were used were homogeneous dry powders containing more than 70% peptide-nitrogen material, except Salmon 3, 4 and 5 with 58%lipids. Because of weak solubility in cell culture medium, these 2.3. Anticancer activity of fish hydrolysates three hydrolysates were discarded. Cod 1–3, Plaice 1–3, Pollack,Salmon 1 and Salmon 2 hydrolysates contained a relatively high sodium chloride content compared to the other hydrolysates.
Two human breast carcinoma cell lines, MCF-7/6 and MDA-MB-231 kindly SEC performed on standard peptides allowed to establish a near provided by Dr. Mareel (Laboratoire de Cance´rologie Expe´rimentale, HoˆpitalUniversitaire, Gand, Belgium) were grown at 37 8C in a 5% CO linear correlation between the retention time (R humidified atmosphere, in DMEM-Ham’s F12 medium (1:1, v:v, Gibco), supple- logarithm of the molecular weight: log (MW) = À0.0996ÁRt mented with 10% heat inactivated (56 8C, 30 min) fetal calf serum (FCS, Dutscher) (min) + 5.52. Peptides contained in cod, plaice, and blue whiting to which were added penicillin 100 U mLÀ1 and streptomycin 100 mg mLÀ1.
hydrolysates showed a large MW distribution ), rangingfrom $100 Da to 7 KDa, indicating the presence of free amino 2.3.2. Antiproliferative activity of fish hydrolysates acids and peptides ranging up to an approximate maximal length In order to perform a screening of the antiproliferative activities of FPH on cancer cells grown in optimal conditions (cell culture medium containing FCS growth factors), a relatively high concentration of fish hydrolysate (1 g LÀ1)was assayed. To avoid pH variation of the cell culture medium during hydro- 3.2. Antiproliferative activity of fish hydrolysates on human lysate solubilization, a 10 g LÀ1 fish hydrolysate stock solution was prepared in PBS 0.1 M pH 7.4, sterile filtered and five fold diluted in cell culture medium(FPH concentration of 2 g LÀ1). The same dilution was prepared replacing FPHstock solution by PBS (control culture medium) or SDS 10% in PBS (lysis As depicted in Blue whiting 3–5, cod, plaice and medium, SDS 2%). Using a multichannel pipettor, 50 mL of each medium were Salmon 2 hydrolysates exerted a significant antiproliferative L. Picot et al. / Process Biochemistry 41 (2006) 1217–1222 Nacl concentration associated to the presence of FPH was limited and appeared insufficient to explain the significant 4.1.2. Presence of anticancer peptides in fish hydrolysates Addition of FPH to a final concentration of 1 g LÀ1 in cell culture medium, compared with the control growth conditions (PBS in cell culture medium), constitutes a supply of nitrogen sources, amino acids, peptides and vitamins that could be used by cancer cells as growth promoting factors, as previously observed in models of gastro-intestinal cell injury and repair .
Conversely, the presence of Blue whiting 3–5, cod, plaice and Salmon 2 hydrolysates in the cell culture medium induced a significant inhibition of cancer cells growth. To identify the nature of molecules responsible for this activity and understand if the process used to prepare FPH could explain and influence the biological activity, a tentative of correlation between activity and FPH composition was made. Blue whiting 3–5 were of particular interest as they contained 96% of peptide-nitrogen material, a content 20% higher than for the rest of the hydrolysates. This observation suggests that the antiproliferative activity could be ND: non-determined. Total percentage can be superior to 100% when a source related to the presence of specific peptides exerting a direct of nitrogen different from proteins is present in the hydrolysate (e.g. urea in siki cytotoxicity on cancer cells, as previously reported for valorphin, a haemoglobin derived peptide , or for an anchovyhydrophobic peptide, able to induce apoptosis in human U937lymphoma cells through the increase of caspases activity activity at 1 g LÀ1. Blue whiting 3–5, containing a very low The hypothesis of a binding competition between fish NaCl concentration and 96% peptide, respectively, induced a peptides and FCS growth factors on cell membrane receptors growth inhibition of 24.5, 22.3 and 26.3% on MCF-7/6, and cannot also be excluded, and fish peptides could then act as 13.5, 29.8 and 29.2% on MDA-MB-231. These values were in antagonists of FCS growth factors receptors. No clear correlation the range of those measured in the presence of etoposide, between degree of hydrolysis and antiproliferative activity was roscovitine or kenpaullone 10À6 M (data not shown) which determined. An identical enzymatic hydrolysis process applied exerted a moderate antiproliferative activity on cancer cells.
to different batches of a given fish source resulted in comparable Antiproliferative activity of FPH were dependent on cell lines global composition and molecular weight profiles as observed for investigated. MDA-MB-231, classed as a highly invasive breast the three cod hydrolysates, the three plaice hydrolysates or Blue cancer cell line, was usually less sensitive to a fish hydrolysate whiting 1–3 hydrolysates. The cod hydrolysates (as well as the treatment than MCF-7/6. This observation is in accordance with plaice hydrolysates), displaying equivalent global composition previous data reported when the two cells lines were grown in and hydrolysis degree, exerted equivalent antiproliferative the presence of antiproliferative thiazolocarbazoles analogues activity. Conversely, in spite of comparable global compositions and molecular weight profiles, Blue whiting 3 exerted a highantiproliferative activity, while Blue whiting 1 and 2 had no activity. This variation of activity remains to be clearly explainedas the only difference in between these hydrolysates is the blue 4.1. Relationship between antiproliferative activity and fish whiting batch, and no modification of the process (temperature, enzyme) can account for the variation in their activity.
4.1.1. Influence of sodium chloride content FPH usually contain a moderate NaCl content due to salting 4.1.3. Presence of other bioactive compounds in fish for conservation or pH adjustements during the pH shift process. The most salted FPH (Plaice 2 and Salmon 1), The lipid quantitative content was not identified as a relevant respectively, contained 21 and 21.4% (w:w) sodium chloride.
marker to predict the antiproliferative activity. For example, the Consequently, presence of FPH at a final concentration of lipid content of all blue whiting hydrolysates was quantitatively 1 g LÀ1 in cell culture medium, compared with the control equivalent but the antiproliferative activity of Blue whiting 3–5 growth conditions, corresponded to a maximal supply of was much greater than that of Blue whiting 1 and 2. This analysis 0.22 g NaCl/L of cell culture medium. Considering that does not exclude that the presence of peculiar bioactive lipids in DMEM and Ham’s F12 are isotonic cell culture media hydrolysates, as well as trace elements of minerals or other (9 g inorganic salts LÀ1, 4750 mg NaCl LÀ1), the increase in bioactive compounds such as fin chondroitine sulfate, or L. Picot et al. / Process Biochemistry 41 (2006) 1217–1222 Fig. 1. Molecular weight distribution of material absorbing at 220 nm in fish hydrolysates. Samples were run as described in Section (a) Cod 1 (full line), Cod 2(dotted line) and Cod 3 (broken line); (b) Blue whiting 4 (full line) and Blue whiting 5 (dotted line); (c) Plaice 1 (full line), Plaice 2 (dotted line), Plaice 3 (broken line);(d) Blue whiting 1 (full line), Blue whiting 2 (dotted line) and Blue whiting 3 (broken line); (e) Salmon 1 (full line) and Salmon 2 (dotted line).
arsenobetaine in plaice could influence the antiproliferative controlled enzymatic hydrolysis of muscle proteins, exert a significant antiproliferative activity on human cancer cell linesin vitro. These preliminary data suggest that FPH could represent an interesting source of anticancer peptides or lipidsto be explored. Preliminary study of the global fish hydrolysate Anticancer molecules isolated from marine organisms composition dit not allow to correlate the antiproliferative belong to diverse structural classes including polyketides, activity to the presence of any peculiar molecules, particularly terpenes, steroids and peptides . These molecules are peptides of defined molecular weight. Moreover, slight usually obtained from fixed animals such as corals, sponges and variations in the hydrolysis process led to high variations in ascidians, which protect themselves from predation by bioactivity, thus confirming the need to accurately control the synthesizing potent cytotoxic molecules. However, fish tissues hydrolysis process to ensure repeatability of FPH bioactivity.
also constitute a potential source of anticancer molecules to be Industrials and scientists searching for bioactive compounds in explored. For example, squalamine, an aminosterol isolated fish hydrolysates are very aware of the rules and regulations to from the liver of the dogfish shark Squalus acanthias , was follow in order to ensure consumer’s safety and efficacy of new demonstrated to be a potent inhibitor of angiogenesis and products. In this view, the demonstration of bioactive properties tumour growth in several animal models Alkylglycer- in in vitro screening tests does by no means prove that fish ols, natural etherlipids abundant in shark liver oil, were recently peptides (or native proteins) exert the same beneficial effects described as inhibitors of tumor vascularization . To our when consumed by humans. Gastric and intestinal protein knowledge, the only anticancer peptide described from a fish digestion generates a huge variety of short chain peptides source is a 440.9 Da anchovy hydrophobic peptide, able to among which only di- and tripeptides are absorbed into induce apoptosis in human U937 lymphoma cells through the intestinal epithelial cells by active transporters located in the increase of caspase-3 and caspase-8 activity The apical membrane of enterocytes . Further studies will be present study demonstrates that some FPH, obtained by necessary to determine whether fish or shellfish ingestion can L. Picot et al. / Process Biochemistry 41 (2006) 1217–1222 [6] LeBlanc JG, Matar C, Valdez JC, LeBlanc J, Perdigon G. Immunomo- dulating effects of peptidic fractions issued from milk fermented withLactobacillus helveticus. J Dairy Sci 2002;85(11):2733–42.
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